Spatiotemporal fluctuations in fluorescence intensity of rhodamine phalloidin-labeled actin filaments

Phalloidin (Ph) is widely used for fluorescent labeling of actin filaments. We observed ADP-actin filaments labeled with rhodamine phalloidin (RhPh) or Alexa488-Ph in vitro and discovered that the fluorescence intensities along the filaments showed a mottled pattern of bright and dark regions. Filaments labeled with substoichiometric RhPh exhibited more significant fluorescence inhomogeneities than those labeled with excess RhPh. Because the quantum yield of Alexa488 fluorescence is hardly affected by the environment, we concluded that the inhomogeneities arise from nonuniform Ph binding density rather than locally inhomogeneous quantum yield of the fluorophores. Simulations assuming random RhPh binding alone partially produced fluorescence inhomogeneities, but the degree of inhomogeneities was significantly smaller than the experimental results. Furthermore, filaments colabeled with RhPh and Alexa488-Ph showed a positive correlation in fluorescence intensities of Rh and Alexa488. Moreover, addition of inorganic phosphate suppressed the fluorescence inhomogeneities and the correlation between the Rh and Alexa488 fluorescence intensities. These results indicated that two mechanisms contribute to the nonuniform binding density of Ph: (i) stochastic binding and (ii) local differences in Ph binding affinity caused by inorganic phosphate-sensitive structural polymorphism of actin filaments. This structural polymorphism may also affect the binding of various actin-binding proteins, contributing to the functional differentiation of actin filaments in vivo. Moreover, those mottled fluorescence patterns dynamically fluctuated over time. These temporal fluorescence fluctuations required glucose and glucose oxidase but were suppressed by Trolox, likely reflecting photophysical properties of fluorophores influenced by oxygen scavengers and triplet-state quenchers. Taken together, we provide new insights into the structural polymorphism of actin filaments.